1. Technical Field
This disclosure relates to cloning vectors. More specifically, phagemid vectors useful in the cloning and expression of foreign genetic information are disclosed.
2. Background of Related Art
Plasmids are extrachromosomal genetic elements capable of autonomous replication within their hosts. Bacterial plasmids range in size from 1 Kb to 200 Kb or more and encode a variety of useful properties. Plasmid encoded traits include resistance to antibiotics, production of antibiotics, degradation of complex organic molecules, production of bacteriocins, such as colicins, production of enterotoxins, and production of DNA restriction and modification enzymes.
Although plasmids have been studied for a number of years in their own right, particularly in terms of their replication, transmissibility, structure and evolution, with the advent of genetic engineering technology the focus of plasmid research has turned to the use of plasmids as vectors for the cloning and expression of foreign genetic information. In its application as a vector, the plasmid should possess one or more of the following properties. The plasmid DNA should be relatively small but capable of having relatively large amounts of foreign DNA incorporated into it. The size of the DNA insert is of concern in vectors based on bacteriophages where packing the nucleic acid into the phage particles can determine an upper limit. The plasmid should be under relaxed replication control. That is, where the replication of the plasmid molecule is not strictly coupled to the replication of the host DNA (stringent control), thereby resulting in multiple copies of plasmid DNA per host cell. The plasmid should express one or more selectable markers, such as the drug resistance markers, mentioned above, to permit the identification of host cells which contain the plasmid and also to provide a positive selection pressure for the maintenance of the plasmid in the host cell. Finally the plasmid should contain a single restriction site for one or more endonucleases in a region of plasmid which is not essential for plasmid replication. A vector as described above is useful, for example, for cloning genetic information, by which is meant integrating a segment of foreign DNA into the vector and reproducing identical copies of that information by virtue of the replication of the plasmid DNA.
The next step in the evolution of vector technology was the construction of so-called expression vectors. These vectors are characterized by their ability not only to replicate the inserted foreign genetic information but also to promote the transcription of the genetic information into mRNA and its subsequent translation into protein. This expression requires a variety of regulatory genetic sequences including but not necessarily limited to promoters, operators, transcription terminators, ribosomal binding sites and protein synthesis initiation and termination codons. These expression elements can be provided with the foreign DNA segment as parts thereof or can be integrated within the vector in a region adjacent to a restriction site so that when a foreign DNA segment is introduced into the vector it falls under the control of those elements to which it is now chemically joined.
Filamentous bacteriophage consist of a circular, single-stranded DNA molecule surrounded by a cylinder of coat proteins. There are about 2,700 molecules of the major coat proteins pVIII that envelope the phage. At one end of the phage particle, there are approximately five copies of each of gene III and VI proteins (pIII and pVI) that are involved in host cell binding and in the termination of the assembly process. The other end contains five copies of each of pVII and pIX that are required for the initiation of assembly and for maintenance of virion stability. In recent years, vectors have been developed and utilized for the display of foreign peptides and proteins on the surface of filamentous phage or phagemid particles.
The display of peptides and proteins on the surface of phage or phagemid particles represents a powerful methodology for selection of rare members in a complex library and for carrying out molecular evolution in the laboratory. The ability to construct libraries of enormous molecular diversity and to select for molecules with predetermined properties has made this technology applicable to a wide range of problems. A few of the many applications of such technology are: i) phage display of natural peptides including, mapping epitopes of monoclonal and polyclonal antibodies and generating immunogens; ii) phage display of random peptides, including mapping epitopes of monoclonal and polyclonal antibodies, identifying peptide ligands, and mapping substrate sites for proteases and kinases; and iii) phage display of protein and protein domains, including directed evolution of proteins, isolation of antibodies and cDNA expression screening.
Vectors have been developed which incorporate DNA from plasmids and bacteriophage. These phagemid vectors are derived by modifications of a plasmid genome containing an origin of replication from a bacteriophage, (e.g. f1, M13, fd) as well as the plasmid origin of replication. Phagemids are useful for the expression of foreign genetic information.
One known phagemid vector is pBluescript II KS+ (pBS II KS+) (Stratagene, La Jolla, Calif.), which is a useful starting point for the construction of the present vector because of its small size and the fact that it contains the colE1 plasmid origin of replication and the phage f1 origin of replication in the desired orientation. The plasmid also carries an ampicillin resistance gene.
Vectors which due to their structures provide enhanced functionality would be desirable.
Novel plasmid vectors capable of replication and expression of foreign genetic information in bacteria, such as, for example, cyanobacterium and E. coli are described herein. These new vectors contain a specific sequence of features after the ColE1 origin but before the f1 origin. Specifically, the present phagemid vector contains, after the ColE1 origin but before the f1 origin, a bacterial transcription terminator, a promoter, a first ribosomal binding site, a first leader sequence and a first cloning region, a second ribosomal binding site, a second leader sequence and a second cloning region. The second cloning region is adapted to receive a gene encoding a polypeptide to be displayed and a nucleotide sequence encoding at least a functional domain of a display protein.
The vectors described herein are constructed through a series of steps which convert a starting vector through a series of intermediate plasmids to the present novel vector which can be used for display of antibody libraries.